Archive for February 9th, 2013

What is gene therapy?

Genes, which are carried on chromosomes, are the basic physical and functional units of heredity. Genes are specific sequences of bases that encode instructions on how to make proteins. Although genes get a lot of attention, it’s the proteins that perform most life functions and even make up the majority of cellular structures. When genes are altered so that the encoded proteins are unable to carry out their normal functions, genetic disorders can result.

Gene therapy is a technique for correcting defective genes responsible for disease development. Researchers may use one of several approaches for correcting faulty genes:

A normal gene may be inserted into a nonspecific location within the genome to replace a nonfunctional gene. This approach is most common.

An abnormal gene could be swapped for a normal gene through homologous recombination.

The abnormal gene could be repaired through selective reverse mutation, which returns the gene to its normal function.

The regulation (the degree to which a gene is turned on or off) of a particular gene could be altered.

How does gene therapy work?

In most gene therapy studies, a “normal” gene is inserted into the genome to replace an “abnormal,” disease-causing gene. A carrier molecule called a vector must be used to deliver the therapeutic gene to the patient’s target cells. Currently, the most common vector is a virus that has been genetically altered to carry normal human DNA. Viruses have evolved a way of encapsulating and delivering their genes to human cells in a pathogenic manner. Scientists have tried to take advantage of this capability and manipulate the virus genome to remove disease-causing genes and insert therapeutic genes.

Target cells such as the patient’s liver or lung cells are infected with the viral vector. The vector then unloads its genetic material containing the therapeutic human gene into the target cell. The generation of a functional protein product from the therapeutic gene restores the target cell to a normal state. See a diagram depicting this process.

Some of the different types of viruses used as gene therapy vectors:

Retroviruses – A class of viruses that can create double-stranded DNA copies of their RNA genomes. These copies of its genome can be integrated into the chromosomes of host cells. Human immunodeficiency virus (HIV) is a retrovirus.

Adenoviruses – A class of viruses with double-stranded DNA genomes that cause respiratory, intestinal, and eye infections in humans. The virus that causes the common cold is an adenovirus.

Adeno-associated viruses – A class of small, single-stranded DNA viruses that can insert their genetic material at a specific site on chromosome 19.

Besides virus-mediated gene-delivery systems, there are several nonviral options for gene delivery. The simplest method is the direct introduction of therapeutic DNA into target cells. This approach is limited in its application because it can be used only with certain tissues and requires large amounts of DNA.

Another nonviral approach involves the creation of an artificial lipid sphere with an aqueous core. This liposome, which carries the therapeutic DNA, is capable of passing the DNA through the target cell’s membrane.

Therapeutic DNA also can get inside target cells by chemically linking the DNA to a molecule that will bind to special cell receptors. Once bound to these receptors, the therapeutic DNA constructs are engulfed by the cell membrane and passed into the interior of the target cell. This delivery system tends to be less effective than other options.

Researchers also are experimenting with introducing a 47th (artificial human) chromosome into target cells. This chromosome would exist autonomously alongside the standard 46 –not affecting their workings or causing any mutations. It would be a large vector capable of carrying substantial amounts of genetic code, and scientists anticipate that, because of its construction and autonomy, the body’s immune systems would not attack it. A problem with this potential method is the difficulty in delivering such a large molecule to the nucleus of a target cell.

Figure 3-B-1. Computer model of base pairing in DNA. In a normal DNA molecule, adenine (A) is paired with thymine (T), guanine (G) is paired with cytosine (C). The uracil (U) of RNA can also pair with adenine (A), since U differs from T by only a methyl group located on the other side of hydrogen bonding.

A DNA molecule has two strands, held together by the hydrogen bonding between their bases. As shown in the above figure, adenine can form two hydrogen bonds with thymine; cytosine can formthree hydrogen bonds with guanine. Although other base pairs [e.g., (G:T) and (C:T) ] may also form hydrogen bonds, their strengths are not as strong as (C:G) and (A:T) found in natural DNA molecules.

The following figure shows an example of base pairing between DNA’s two strands.

Figure 3-B-2. Schematic drawing of DNA’s two strands.

Due to the specific base pairing, DNA’s two strands are complementary to each other. Hence, the nucleotide sequence of one strand determines the sequence of another strand. For example, in Figure 3-B-2, the sequence of the two strands can be written as

5′ -ACT- 3′

3′ -TGA- 5′

Note that they obey the (A:T) and (C:G) pairing rule. If we know the sequence of one strand, we can deduce the sequence of another strand. For this reason, a DNA database needs to store only the sequence of one strand. By convention, the sequence in a DNA database refers to the sequence of the 5′ to 3′ strand (left to right).

There is a major difference between DNA polymerase and RNA polymerase: the RNA polymerase can synthesize a new strand whereas the DNA polymerase can only extend an existing strand. Therefore, to synthesize a DNA molecule, a short RNA molecule (~ 5 – 12 nucleotides) must be synthesize first by a special enzyme. The initiating RNA molecule is known as a primer, and the enzyme is called primase.

In addition to DNA polymerase and primase, DNA replication requires helicase and single strand binding protein (SSB protein). The role of helicase is to unwind the duplex DNA. SSB proteins can bind to both separated strands, preventing them from annealing (reconstitution of double-stranded DNA from single strands).

The replication mechanisms in both bacteria and eukaryotes are similar. However, eukaryotic DNA polymerases do not contain a subunit similar to the E. colib subunit. They use a separate protein called proliferating cell nuclear antigen (PCNA) to clamp the DNA.

Figure 7-B-2. Structure of PCNA which is formed by three identical subunits. PDB ID = 1AXC.

DNA polymerases can extend nucleic acid strands only in the 5′ to 3′ direction. However, in the direction of a growing fork, only one strand is from 5′ to 3′. This strand (the leading strand) can be synthesized continuously. The other strand (the lagging strand), whose 5′ to 3′ direction is opposite to the movement of a growing fork, should be synthesized discontinuously.

Figure 7-B-3. Steps in the synthesis of the lagging strand.

(a) Comparison between the leading strand and the lagging strand.

(b) The primase first synthesizes a new primer which is about 10 nucleotides in length. The distance between two primers is about 1000-2000 nucleotides in bacteria, and about 100-200 nucleotides in eukaryotic cells.

(c) DNA polymerase elongates the new primer in the 5′ to 3′ direction until it reaches the 5′ end of a neighboring primer. The newly synthesized DNA is called an Okazaki fragment.

(d) In E. coli, DNA polymerase I has the 5′ to 3′ exonuclease activity, which is used to remove a primer.

(e) DNA ligase joins adjacent Okazaki fragments.

The whole lagging strand is synthesized by repeating steps (b) to (e).

Mendel then crossed a pure & a hybrid from his F2 generation; known as an F2 or test cross

Trait – Plant Height

Alleles – T tall, t short

F2 cross TT x Tt

F2 cross tt x Tt

T

t

T

t

T

TT

Tt

t

Tt

tt

T

TT

Tt

t

Tt

tt

genotype – TT, Tt

genotype – tt, Tt

phenotype – Tall

phenotype – Tall & short

genotypic ratio– 1:1

genotypic ratio– 1:1

phenotypic ratio– all alike

phenotypic ratio– 1:1

50% (1/2) of the offspring in a test cross showed the same genotype of one parent & the other 50% showed the genotype of the other parent; always a 1:1 ratio

Problems:Work the P1, F1, and both F2 crosses for all of the other pea plant traits & be sure to include genotypes, phenotypes, genotypic & phenotypic ratios.

Mendel also crossed plants that differed in two characteristics (Dihybrid Crosses)
such as seed shape & seed color

In the P1 cross, RRYY x rryy, all of the F1 offspring showed only the dominant form for both traits; all hybrids, RrYy

Traits: Seed Shape & Seed Color

Alleles: R round Y yellow
r wrinkled y green

P1 Cross: RRYY x r r yy

ry

Genotype:

RrYy

RY

RrYy

Phenotype:

Round yellow seed

Genotypic ratio:

All alike

Phenotypic ratio:

All Alike

When Mendel crossed 2 hybrid plants (F1 cross), he got the following results

Traits: Seed Shape & Seed Color

Alleles: R round Y yellow
r wrinkled y green

F1 Cross: RrYy x RrYy

RY

Ry

rY

ry

RY

RRYY

RRYy

RrYY

RrYy

Ry

RRYy

RRyy

RrYy

Rryy

rY

RrYY

RrYy

r rYY

r rYy

ry

RrYy

Rryy

r rYy

r ryy

Genotypes

Genotypic Ratios

Phenotypes

Phenotypic Ratios

RRYY

1

Round yellow seed

9

RRYy

2

RrYY

2

RrYy

4

RRyy

1

Round green seed

3

Rryy

2

r rYY

1

Wrinkled yellow seed

3

r rYy

2

r ryy

1

Wrinkled green seed

1

Problems:Choose two other pea plant traits and work the P1 and F1 dihybrid crosses. Be sure to show the trait, alleles, genotypes, phenotypes, and all ratios.

Results of Mendel’s Experiments:

Inheritable factors or genesare responsible for all heritable characteristics

Phenotype is based on Genotype

Eachtrait is based on two genes,one from the mother and the other from the father

True-breedingindividuals are homozygous ( both alleles) are the same

Law of Dominance states that when different alleles for a characteristic are inherited (heterozygous), the trait of only one (the dominant one) will be expressed. The recessive trait’s phenotype only appears in true-breeding (homozygous) individuals

Trait: Pod Color

Genotypes:

Phenotype:

GG

Green Pod

Gg

Green Pod

gg

Yellow Pod

Law of Segregation states that each genetic trait is produced by a pair of alleles which separate (segregate) during reproduction

Rr

R

r

Law of Independent Assortment states that each factor (gene) is distributed (assorted) randomly and independently ofone another in the formation of gametes

RrYy

RY

Ry

rY

ry

Other Patterns of Inheritance:

Incomplete dominance occurs in the heterozygous or hybrid genotype where the 2 alleles blend to give a different phenotype